Project Summary Mammalian prions, which cause fatal neurodegenerative diseases in humans and livestock animals, are unorthodox infectious agents that replicate by the autocatalytic conformational change of the host prion protein (PrPC) into a pathogenic isoform (PrPSc). Taking a reductionist biochemical approach, we made the surprising discovery that endogenous cofactors such as the membrane lipid phosphatidylethanolamine (PE) are required to produce infectious PrPSc molecules. For example, autocatalytic PrPSc molecules formed with a combination of recombinant PrP and PE substrates display a specific infectivity >105-fold greater than that of similar autocatalytic PrPSc molecules formed from PrP alone. We have taken advantage of this specific information to produce, for the first time, sufficient quantities of fully infectious, isotopically labeled, and conformationally homogeneous recombinant prions to perform high-resolution solid state (ss) NMR analysis of PrPSc structure. Here, our collaborative team proposes to use ssNMR, electron microscopy, and mass spectrometry analysis to elucidate the important structural elements that lead to infectivity. ssNMR will be used to determine and compare the secondary structure maps of infectious and non-infectious PrPSc molecules. This work will help identify and characterize specific infectivity-associated domains as well as crucial NMR residue-peak assignments. Together with constraints from electron microscopy and mass spectrometry analysis to identify the position of cut points using digestion by proteinase K, these data will eventually enable structural determination of the entire PrPSc molecule. We will also use ssNMR to determine precisely the structural mechanism by which PE induces the infectious prion conformation. Finally, we will use 15N-13C transferred- echo double resonance (TEDOR) to determine the symmetry pattern of PrPSc subunits in isotopically mixed samples of infectious recombinant prions. These data will allow us to discriminate between competing b-solenoid and in-register b-sheet quaternary structure models of infectious PrPSc molecules. Overall, this proposal is a critical step towards determining the full high-resolution structural determination of infectious mammalian prions as well as the structural mechanism of prion infectivity.